Method of use of reusable sample holding device permitting ready loading of very small wet samples

ABSTRACT

A method for using a reusable sample-holding device for readily loading very small wet samples for observation of the samples by microscopic equipment, in particular in a vacuum environment. The method may be used with a scanning electron microscope (SEM), a transmission electron microscope (TEM), an X-ray microscope, optical microscope, and the like. For observation of the sample, the method provides a thin-membrane window etched in the center of each of two silicon wafers abutting to contain the sample in a small uniform gap formed between the windows. This gap may be adjusted by employing spacers. Alternatively, the thickness of a film established by the fluid in which the sample is incorporated determines the gap without need of a spacer. To optimize resolution each window may have a thickness on the order of 50 nm and the gap may be on the order of 50 nm.

RELATED APPLICATIONS

This is a division of U.S. patent application Ser. No. 12/365,698,originally filed as Reusable Sample Holding Device Permitting ReadyLoading of Very Small Wet Samples on Feb. 4, 2009 U.S. Pat. No.8,059,271 by Marsh et al., and incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

Under paragraph 1(a) of Executive Order 10096, the conditions underwhich this invention was made entitle the Government of the UnitedStates, as represented by the Secretary of the Army, to an undividedinterest therein on any patent granted thereon by the United States.This and related patents are available for licensing to qualifiedlicensees. Please contact Bea Shahin at 217 373-7234.

BACKGROUND

Nanotechnology of materials, related microbiological means ofmanipulation, and synthetic biology require a means to directly observenanoscale interactions and classify nano-material morphologicalproperties in solution on a small scale. More specifically the lack ofparticle and colloid size descriptors and in situ nanoscale imagingtechniques are detrimental to advancing the state of the art because itinhibits the ability to verify the reproducibility of nano-fluidpreparation necessary for research groups to compare properties.

Using and relying only on more conventional imaging techniques such as atransmission electron microscope (TEM) and a scanning electronmicroscope (SEM) raises several concerns. TEM and SEM samples are driedand exposed to vacuum before being imaged which changes the observedfundamental properties of the sample. Further, when using a wet cellwith a TEM, images should be taken immediately since the energy of thebeam quickly de-hydrates the small amount of fluid within the sample.Using conventional techniques, this does not lend itself to accurateparticle and agglomerate size characterization much less quantificationof other physio-chemical properties.

Besides standard TEM use, the current suite of options available toobserve objects at the nanoscale in situ includes the electroncryo-microscope, dynamic light scattering (DLS), small angle x-rayscattering (SAXS), small angle neutron scattering (SANS), and othersadapted for more specific applications. The electron cryo-microscopepresents drawbacks to drying similar to the TEM and SEM as the samplemust be flash frozen into vitreous ice. The procedure distorts the imageas well as limiting sample thickness that may be addressed. Moreover, itis undesirable to freeze a sample because the properties of thesolution, including species interaction and separation, change. Dynamiclight scattering is a very reliable way to determine particle size. DLSworks with a broad range of materials in most instances. However, DLSdoes not work well with non-spherical species such as carbon nanotubesor DNA. Like DLS, small-angle X-ray scattering (SAXS), and small-angleneutron scattering (SANS) are broadly used in research and industry.SAXS has a rather low observable size limit, not able to detect thelength of most carbon nanotubes (CNTs), and is best suited for analyzingthe surface and bulk properties of larger samples. SANS shares some ofthe undesirable traits of SAXS and adds the threat of sample damage vianeutron-particle interaction. This degrades observation of the extent ofcolloidal agglomeration, for example.

Existing methods, devices, developments and publications are inherentlylimited. U.S. Pat. No. 4,071,766, to Kalman et al., employs afilm-sealed micro-chamber to hold a wet sample. There are bores or pipesto introduce fluid to this micro-chamber. However, the large chamber orgap height (the length that the electron beam needs to traverse) makesit impractical for use in transmission electron microscopy. For mediumhigh-voltage TEMs, typical sample thickness which is transparent toelectron beam is below 700 nm.

Fukami et al. use a design similar to Kalman et al. but with an assemblycomprising multiple parts to form the chamber instead of one integralpart. A. Fukami and K. Fukushima, Proc. Eighth European Congress onElectron Microscopy, Budapest, pp. 71-80, 1984. A Fukami, K. Fukushimaand N. Kohyama, Observation Techniques for Wet Clay Minerals UsingFilm-Sealed Environmental Cell Equipment Attached to High-ResolutionElectron Microscope, In: Microstructure of Fine-Grained Sediments fromMud to Scale, R. H. Bennett, W. R. Bryant, M. H. Hulbert (eds.), NewYork: Springer-Verlag, pp. 321-331, 1991. Carbon films supported bycopper grids are used as the sealing film. Again, the spacing betweenthe films is large. The film is easily broken which can result inextreme damage to microscopy equipment.

U.S. Pat. No. 5,406,087, to Fujiyoshi et al., describes aspecimen-holding device that is quite similar to the Fukami et al.design. Two polymer films are pressed together directly by backingcopper grids and the samples are trapped between the films. The polymerfilms and the thin copper grids tend to deform making it impossible toform thin liquid films over relatively large areas. Further, the film iseasily broken which can result in extreme damage to microscopyequipment. Moreover, the design does not have any element to control thesize of the gap and requires accurate alignment of the grids, which isdifficult.

Williamson et al. construct a wet cell by gluing two Si₃N₄-coatedsilicon wafers face to face. M. J. Williamson, R. M. Tromp, P. M.Vereecken, R. Hull, and F. M. Ross, Dynamic Microsocopy of NanoscaleCluster Growth at the Solid-Liquid Interface, Nature Materials, Vol. 21,pp. 532-536, August 2003. Each wafer has a Si₃N₄ thin film membranewindow formed by selective etching. The wafers are adhered to oneanother around the edges and a gap between the wafers is created with adeposited SiO₂ spacer element. Liquid is loaded into the cell throughports on one wafer. The cell is sealed by gluing sapphire plates overthe ports. This invention involves a tedious and time-consuming processinvolving multiple gluing and curing steps. Further, the glue tends toenlarge the gap. This device does not provide for a sample that is thinenough for observation of desired characteristics.

The following U.S. patents suffer from similar drawbacks as discussedabove: U.S. Pat. No. 7,476,871 to Chao et al.; U.S. Pat. No. 7,304,313to Moses et al.; U.S. Pat. No. 7,253,418 to Moses et al.; U.S. Pat. No.7,230,242 to Behar et al.; U.S. Pat. No. 7,219,565 to Fischione et al.;U.S. Pat. No. 6,992,300 to Moses et al.; U.S. Pat. No. 6,989,542 toMoses et al.; U.S. Pat. No. 5,412,211 to Knowles; U.S. Pat. No.5,362,964 to Knowles et al.; U.S. Pat. No. 5,097,134 to Kimoto et al.;and U.S. Pat. No. 4,705,949 to Grimes et al.

A process capable of allowing one to analyze in situ samples of a broadnature mitigates many of the above limitations of conventional methods.Further, it would be advantageous to have a sample holding device withthe following characteristics: self-aligning windows on holder piecesand wafers, thus providing for ease of assembling the sample in theholder; a controllable sample volume (gap) thickness via modifyingspacer (integral to the wafer or separate) thickness; uniform samplevolume thickness; improved spatial resolution resulting from reduced gap(sample volume) thickness; improved resolution resulting from use ofultra-thin (<100 nm) membranes as windows, in particular Si₃N₄-coatedmembranes; durable and reliable sealing of the sample combined with awindow pattern that provides reinforcement resulting in safe use invacuum environments.

A reliable readily implemented in situ imaging technique embodied inselect embodiments of the present invention has the abovecharacteristics and allows direct examination of nano-fluid sampleswithout affecting basic characteristics, such as nano-particledispersion.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elevation view of a vertical slice through the center of asample holding device used in the present invention.

FIG. 2A is a perspective top view of major parts a sample holding deviceused in the present invention depicting relative size.

FIG. 2B is FIG. 2A with the silicon wafers installed in the bottomportion of a sample holding device used in the present invention, alsodepicting relative size.

FIG. 3 is a perspective view of major parts of the sample holding deviceof FIGS. 1, 2A and 2B in an expanded vertical parts diagram as used inthe present invention.

FIG. 4 is a photo of carbon nanotubes (low-Z materials) in water, takenusing a method of the present invention.

DETAILED DESCRIPTION

Select embodiments of the present invention comprise a method for usingreusable sample-holding devices for readily loading very small wetsamples for observation of the samples, in particular in a vacuumenvironment. Select embodiments of the present invention may be usedwith an electron microscope, such as a transmission electron microscope(TEM), an x-ray microscope, optical microscope, and the like. Selectembodiments of the present invention provide a small volume formedbetween two silicon wafers for holding the sample via use of a “spacer,”such as a washer, for separating the wafers. This volume may beadjustable by employing spacers of different thicknesses. The spacersmay be integral with one side of one or both of the silicon wafers ormay be separate pieces. Alternatively, the thickness of the filmestablished by the fluid in which the sample exists may determine thevolume (gap) without need for use of a spacer.

Each silicon wafer is provided with a transparent thin-film siliconnitride (Si₃N₄) membrane that serves as a transparency affixed over anopening (window) in a center section in each silicon wafer. A portion ofthe center of each wafer is removed, e.g. by etching, to provide foraffixing the membrane on one side of the center portion of each wafer,the membrane, e.g. one of Si₃N₄ less than about 100 nm thick, preferablyabout 50 nm thick. Thin membranes assure high resolution although at theexpense of being somewhat fragile at a thickness less than 100 nm. Inselect embodiments of the present invention, high vapor pressure fluidis held in the small volume, typically a uniform gap established betweenthe two silicon wafers by the spacer or spacers. In select embodimentsof the present invention, a uniform gap may be maintained by inserting awasher between the silicon wafers and centering the washer on the window(opening) in the center of each wafer.

In select embodiments of the present invention, a sample may be observedthrough either side of the transparent thin-film membranes incorporatedin each of the silicon wafers although the top section of the device maybe specifically configured to interface with specialized instrumentssuch as a TEM and thus optimize viewing through a configuration integralwith the top of the device. In select embodiments of the presentinvention, the openings (windows) in similarly shaped silicon wafers areautomatically aligned during assembly since the windows are centered ineach of the silicon wafers and the wafers are the same shape and length(along their perimeter) as a depression in the lower part (i.e., thesample holding device) for retaining the silicon wafers and spacer orspacers.

Select embodiments of the present invention successfully and reliablyseal fluid, typically a liquid, between the silicon wafers through useof seals such as O-rings. Further, select embodiments of the presentinvention are compatible with a high vacuum environment as is found inelectron and x-ray microscopy.

Select embodiments of the present invention permit quick and easystudies of samples placed in a fluid. In select embodiments of thepresent invention, maintaining a consistent gap between two identical“windowed” silicon wafers assures uniform thickness of the sample whileproviding very thin transparent membranes as the window material ensureshigh resolution. Select embodiments of the present invention further maybe employed with “lab on a chip” designs that incorporate electrodes andfluidic elements for detecting and recording changes that may be imposedon the sample during viewing, such as temperature and pressure changes,electrical and magnetic field changes, and the like.

Select embodiments of the present invention are employed by sandwichinga fluid containing the sample of interest between two silicon wafersincorporating thin-film membrane windows in the center of the side ofeach wafer abutting the fluid containing the sample. The thin-filmmembrane preferably comprises Si₃N₄. Thin-film membranes are availablefrom Structure Probe, Inc., P.O. Box 656, West Chester, Pa. 19380.Preferably the thin-film membrane “windows” are fabricated by etchingthe center of a silicon wafer of original thickness about 200 μm to athickness less than about 100 nm and coating the resultant thinnedportion of the etched section with Si₃N₄ to create a thin-membrane Si₃N₄window for observing samples immersed in a fluid or wet samples.Preferably this thin-membrane Si₃N₄ window is of a thickness about 50nm.

Select embodiments of the present invention employ a two-piececonfiguration of a top and bottom section that abut upon final securingof a sample. The bottom section further comprises an “impression” withan opening in its center that coincides with the openings (windows) ofthe two silicon wafers separated by one or more spacers to be placedtherein. The impression holds and “indexes” the spaced apart wafers sothat the center-located windows of the wafers are aligned with eachother and the openings in the top and bottom sections. The impressionalso incorporates a circular slot for each of an inner and outer O-ring.These O-rings are secured upon affixing the top section to the bottomsection. The opening (window) in the center of each silicon wafer iscovered with a transparent thin-film membrane, e.g., a membrane of Si₃N4that is affixed to the side of the wafer abutting the sample. Preferablythe thin-film membranes are of a thickness of less than about 100 nm.The windows are aligned to permit viewing of the sample from either thetop or the bottom of the holder device although select embodiments ofthe present invention embodiments are specifically configured tointerface with existing systems for viewing only from the top. In selectembodiments of the present invention, various materials, sample holdersizes, and thicknesses may be employed to address specific userrequirements. In select embodiments of the present invention, a wafer“sandwich” is placed in a sample holder comprised at least in part ofnon-magnetic material and incorporates O-rings that create a sealholding a vacuum for the fluid containing the sample placed in thedevice. Emplacing and securing a wet sample using an embodiment of thepresent invention takes 10-15 minutes. For select embodiments of thepresent invention, no alteration to the viewing instrument, such as amicroscope or a TEM unit, is necessary.

A method to allow microscopic observation of a sample comprises:acquiring and sizing the sample to be observed; inserting the sample ina fluid; positioning the top and bottom pieces for the holder device forcleaning; cleaning the top and bottom pieces with a suitable solventsuch as isopropyl alcohol; placing a first O-ring in the slot in thebottom piece, placing a first silicon wafer incorporating a first Si₃N₄thin-film membrane in its center in the impression in the bottom piece;placing fluid containing the sample of interest on the center of thefirst silicon wafer with a pipette or similar device; placing a spacercentered over the center of the first silicon wafer if neither the firstor second silicon wafers have an integral spacer or an integral spacerof sufficient depth; placing a second silicon nitride waferincorporating a second window, the second window abutting the fluidcontaining the sample in the impression, the second silicon waferself-aligning in the impression with the first silicon wafer; placing asecond inner O-ring in a slot encircling the window in the top piece;placing an outer O-ring in a slot in the top piece, the outer O-ringcapable of encircling the entire impression of the bottom piece; placingthe top piece with inserted O-rings over the bottom piece, aligningscrew heads in the bottom piece with slots in the top piece; rotatingthe top piece to align it under the screw heads and tightening the screwheads as necessary for retention of the seals made by the O-rings, andemplacing the resultant sample holding device in proximity to theviewing device, indexing the holding device with the viewing device asnecessary.

Refer to FIG. 1 depicting a vertical slice longitudinally through anapparatus employed with select embodiments of the present invention,specifically detailing the center section. The “body” (frame) of thesample holder 100 comprises two parts, a bottom piece 101 and a toppiece 102 configured to affix to the bottom piece 101 upon finalassembly of the sample holder 100. The bottom piece 101 is etchedcompletely through its center creating a centered opening 101D andincludes an etched rectangular slot 103 centered over the opening 101D.The rectangular slot 103 is configured to contain silicon wafers 104,105 and “indexes” the wafers 104, 105 in the sample holder 100 via amatching fit of the wafers 104, 105 to the sidewalls 101A of the etchedrectangular slot 103. Prior to final assembly of the sample 112 in thesample holder 100, a first O-ring 109 is placed in a circular slot 101Bcentered within the etched rectangular slot 103. A first silicon wafer104, etched to configure a thin-membrane Si₃N₄-coated window 104A in thecenter of one side of the silicon wafer 104, is then placed within therectangular slot 103 with the thin-membrane window 104A facing up. Aspacer 106, such as a thin washer, for maintaining a pre-specified gapd, is placed over the bottom piece 104, preferably centered on theopening 101D and the thin-membrane Si₃N₄-coated window 104A.Alternatively, the spacer 106 may be incorporated in either or both ofthe silicon wafers 104, 105 by etching or the like to meet a user'srequirements. The sample 112 is placed upon the Si₃N₄-coated window 104Aand a second silicon wafer 105 configured like the first silicon wafer104 in size and dimension to include a Si₃N₄-coated window 105A centeredthereon is placed, with the Si₃N₄-coated window 105A abutting the sample106, upon the spacer 106 in the rectangular slot 103. A second O-ring107 is placed in a slot 101C circumscribing the rectangular slot 103. Athird O-ring 108 with a diameter similar to the first O-ring 109 isplaced in a slot 102B in the top piece 102, the top piece 102incorporating an opening 102D similar to that of the bottom piece 101and aligned therewith upon final assembly of the sample holder 100. Thetop piece 102 is affixed to the bottom piece 101 via suitable fastenersinserted in threaded holes 110, compressing all O-rings 107, 108, 109 toform a seal suitable for operation in a vacuum. The sample holder 100may incorporate a tab 114 incorporating a hole 114A for indexing inobservation equipment, such as an SEM or a TEM, or for hanging on a hookin a storage container.

Refer to FIG. 2A, a perspective top view of major parts of analternative embodiment of the sample holder 100 of FIG. 1, furtherdepicting relative size compared to a dime 210. The top (abutting) sideof the bottom piece 101 and the bottom (abutting) side of the top piece102 is shown, both top and bottom 101, 102 pieces differing from theembodiment of FIG. 1 in that the large O-ring 107 is incorporated in theslot 202 of the top piece 102 in the embodiment of FIG. 2A. The slots201 in the top piece 102 are configured to permit rotating the top piece102 once fasteners (machine screws) 203 are loosened. To install the toppiece 102, the wide part of the slots 201 is placed over the fasteners203 and then rotated to the narrow part of the slot 201. The fasteners203 are then torqued to bring the bottom of the top piece 102 in contactwith the top of the bottom piece 102, compressing the O-rings 107, 108,109 in the process. This slot configuration 201 further facilitates bothplacement and removal of samples 112 quickly and easily withoutpotential for losing fasteners (e.g., machine screws) 203 that otherwisewould need to be completely removed to assemble or disassemble thesample holder 100. In FIG. 2A, the silicon wafers 104, 105 are shownseparately from the sample holder 100 for perspective. A thumbnail slot204 is provided in the bottom piece 101 to facilitate removal of thesilicon wafers 104, 105 and any spacer(s) 106 upon disassembly of thesample holder 100.

Refer to FIG. 2B, a perspective top view of major parts of analternative embodiment of the sample holder 100 of FIG. 1, depicting thesilicon wafers 104, 105 installed in the bottom piece and furtherdepicting relative size compared to a dime 210.

Refer to FIG. 3, a perspective view of major parts of a selectembodiment of the sample holder in an expanded vertical parts diagram.The top side of the top piece 102 is shown with a raised portion 301with opening 301A to facilitate use with specific types of observationequipment such as a TEM. The large O-ring 107 is shown to fit as in theconfiguration of FIGS. 2A, 2B and shows the difference in thicknessbetween the top 102 and bottom piece 101 where abutting is apparent.

Materials employed for various components of select embodiments of thesample holder may be pre-specified by a user. For example, non-magneticmaterials such as certain aluminum alloys, phosphor or beryllium bronzesand like relatively light materials, are preferred for the top 102 andbottom 101 pieces, the spacer 106 (preferably beryllium if a separatespacer 106 is employed and not an etched portion of the silicon wafers104, 105), as well as the fasteners 203, with non-magnetic titanium apreferable material for use in at least some of the components.

As to typical sizing of the various elements in select embodiments ofthe sample holder, the depth of the assembled sample holder 100 may beon the order of 3 mm, the silicon wafers 104, 105 may be on the order of200 μm in overall thickness, the spacer 106 may be on the order of about50 nm, the thin-membrane Si₃N₄-coated windows 104A, 105A on the order of50 nm or less depending on the strength required, the thin-membraneSi₃N₄-coated windows 104A, 105A nominally 500 μm on a side for a squarethin-membrane Si₃N₄-coated window 104A, 105A.

Select methods of the present invention may be used to observe fluid(liquid, gas, or both) with a TEM, SEM, X-ray microscope or opticalmicroscope. Other applications include incorporating nano-fluidics inthe sample holder 100 or electrodes on one or both silicon wafers 104,105 to study nano-fluidics or electrochemical processes underhigh-resolution electron microscopes, in particular TEMs. This wouldpermit observing activity of nano-sized species or biological processesin real time.

Refer to FIG. 4, depicting a photo taken of carbon nanotubes (low-Zmaterials) in water by employing a method of the present invention. Thephoto demonstrates that the total thickness of the sample 106 andthin-membrane Si₃N₄ windows 104A, 105A is sufficient for manyapplications. These thin-membrane Si₃N₄ windows 104A, 105A are strong,virtually eliminating the possibility of damage to expensive microscopesshould they burst under a vacuum. Select methods of the presentinvention are relatively inexpensive to implement because everything butthe silicon wafers 104, 105 is reusable, and in some conditions, eventhe wafers 104, 105 may be reusable. Sample holders used withembodiments of the present invention may be reused many times and arenot prone to degradation or structural failure.

The sample holder 100 is easy to assemble using select methods of thepresent invention, with self alignment mechanisms for the wafers 104,105 and their centered thin-membrane Si₃N₄-coated windows 104A, 105A andholder windows 101D, 102D. The gap, d, between the thin-membraneSi₃N₄-coated windows 104A, 105A is controllable, an extremely importantadvantage for employment with a TEM. Image quality is a function oftotal sample thickness, including the two thin-membrane Si₃N₄-coatedwindows 104A, 105A and the fluid containing the sample in the gap, d.Select methods of the present invention facilitate very thin gap sizes,d, e.g., on the order of 50 nm or less, and inherently yield highresolution.

The abstract of the disclosure is provided to comply with the rulesrequiring an abstract that will allow a searcher to quickly ascertainthe subject matter of the technical disclosure of any patent issued fromthis disclosure. (37 CFR §1.72(b)). Any advantages and benefitsdescribed may not apply to all embodiments of the invention.

While the invention has been described in terms of some of itsembodiments, those skilled in the art will recognize that the inventioncan be practiced with modifications within the spirit and scope of theappended claims. For example, although the method is described inspecific examples for holding a small volume wet sample as a very thinfilm for observation in a vacuum environment such as present withdevices such as a TEM, select methods of the present invention may beused for any type of testing or monitoring and thus may be useful insuch diverse applications as laboratory analysis, structural monitoring,remediating, environmental intervention, industrial processing, and thelike. Structure monitored or tested may be of any type ranging fromnaturally occurring to manmade. In the claims, means-plus-functionclauses are intended to cover the structures described herein asperforming the recited function and not only structural equivalents, butalso equivalent structures. Thus, although a nail and a screw may not bestructural equivalents in that a nail employs a cylindrical surface tosecure wooden parts together, whereas a screw employs a helical surface,in the environment of fastening wooden parts, a nail and a screw may beequivalent structures. Thus, it is intended that all matter contained inthe foregoing description or shown in the accompanying drawings shall beinterpreted as illustrative rather than limiting, and the inventionshould be defined only in accordance with the following claims and theirequivalents.

1. A method facilitating microscopic observation of a sample,comprising: providing a sample holding device, comprising: a first waferhaving a length, width and depth and incorporating a transparentmembrane in a first side of said first wafer, said first sideestablished by said length and said width of said first wafer; a secondwafer having a length, width and depth and incorporating a transparentmembrane in a first side of said second wafer, said first sideestablished by said length and said width of said second wafer, whereinsaid second wafer is approximately the same size and geometry as saidfirst wafer; a first component having a length, width and depth, saidfirst component having an opening perpendicular to and through the planeof said first component established by the length and width of saidfirst component, said first component further comprising threadedopenings in a first pattern about the perimeter of said first component,said first component further configured to incorporate both said firstand second wafers abutted along their respective length and width so asto be parallel to each other and said plane of said first component, atopmost of said first and second wafers at or below the top of saidplane of said first component, wherein said first component indexes therelative position of said first and second wafers, said first and secondwafers abutting to contain said sample in a uniform gap formed betweensaid transparent membranes, a second component having a length, widthand depth, said second component having an opening perpendicular to andthrough a plane of said second component established by said length andwidth of said second component, said second component incorporating animpression approximately centered in said second component, said secondcomponent further comprising first slots in a second pattern about theperimeter of said second component, said second pattern movablyalignable with said first pattern to allow threaded fasteners to bealigned with said threaded opening in said first component to affix tosaid first component to secure said sample in said sample holdingdevice; at least one seal in operable communication with said first andsecond components; and at least one fastener incorporating at least ahead and threaded shank compatible with said threaded openings, forremovable affixing said first component to said second component; andperforming the following steps for loading each said sample: a.positioning said first and second wafers of said sample holding devicefor cleaning; b. cleaning said first and second wafers; c. placing afirst inner O-ring in a first slot in said second component, said secondcomponent oriented to be the bottom of said sample holding device; d.placing said first wafer incorporating a first transparent membrane insaid impression of said second component, said impression approximatelycentered in said second component; e. placing said sample on the centerof said first wafer; f. insuring spacing between said first wafer andsaid second wafer to accommodate said sample; g. placing said secondwafer incorporating a second transparent membrane, the secondtransparent membrane abutting said sample in said impression, saidsecond wafer self-aligning in said impression with said first wafer; h.placing a second inner O-ring in a second slot encircling saidtransparent membrane in said first component; i. placing an outer O-ringin a third slot in said second component, said outer O-ring encirclingsaid impression of said second component; j. placing said firstcomponent over said second component with inserted said second inner andsaid outer O-rings, aligning said fastener heads in said secondcomponent with four slots in said first component; k. rotating saidfirst component to align it under said fastener heads; l. establishing anon-fluid flowing chamber by tightening said means for fastening, m.placing said sample holding device in proximity to a viewing device andindexing said holding device with said viewing device, and n. repeatingsteps a through m for each additional said sample.
 2. The method ofclaim 1 providing said sample as a wet sample.
 3. The method of claim 2providing said wet sample at a thickness of about 50 nm or less.
 4. Themethod of claim 1 providing said wafers as silicon wafers.
 5. The methodof claim 4 providing each said silicon wafer at a thickness of about 200μm, wherein said transparent membranes are fabricated in said siliconwafer by at least etching said silicon wafer.
 6. The method of claim 1providing said transparent membranes as thin-film Si₃N₄-coatedmembranes.
 7. The method of claim 6 providing said thin-filmSi₃N₄-coated membranes at less than about 100 nm thick.
 8. The method ofclaim 6 providing said thin-film Si₃N₄-coated membranes at about 50 nmthick.
 9. The method of claim 1 providing a first slot incorporated insaid first component for a first one of said at least one seal, saidfirst seal being a first O-ring in operable communication with at leastsaid first and second components, said first O-ring captured in at leastsaid first slot.
 10. The method of claim 1 providing said secondcomponent incorporating a second slot for a first one of said at leastone seals, said first seal provided as a first O-ring in operablecommunication with at least said first and second components, said firstO-ring captured in at least said second slot.
 11. The method of claim 1providing said first component incorporating a third slot for a secondone of said at least one seals, said second seal provided as a secondO-ring in operable communication with at least said first wafer and saidfirst component, said second O-ring captured in at least said thirdslot.
 12. The method of claim 1 providing said second componentincorporating a fourth slot for a third one of said at least one seals,said third seal provided as a third O-ring in operable communicationwith at least said second wafer and said second component, said thirdO-ring captured in at least said fourth slot.
 13. The method of claim 1providing said fasteners as machine screws.
 14. A method facilitatingmicroscopic observation of a sample, comprising: providing a sampleholding device, comprising: first means for viewing having a length,width and depth for incorporating a transparent means in one side ofsaid first means for viewing, said side established by said length andsaid width of said first means for viewing; a second means for viewinghaving a length, width and depth and incorporating a transparent meansin one side of said second means for viewing, said side established bysaid length and said width of said second means for viewing, whereinsaid second means for viewing is approximately the same size andgeometry as said first means for viewing; a first component having alength, width and depth, said first component having an openingperpendicular to and through the plane of said first componentestablished by the length and width of said first component, said firstcomponent further configured to incorporate both said first and secondmeans for viewing abutted along their respective length and width so asto be parallel to each other and said plane of said first component, atopmost of said first and second means for viewing at or below the topof said plane of said first component, wherein said first componentindexes the relative position of said first and second means forviewing, said first and second means for viewing abutting each other tocontain said sample in a uniform gap formed between said first andsecond means for viewing, a second component having a length, width anddepth, said second component; and performing the following steps forloading each said sample: a. positioning said first and second means forviewing to allow cleaning thereof; b. cleaning said first and secondmeans for viewing; c. placing a first inner means for sealing in a firstslot in said second component, said second component oriented to be thebottom of said sample holding device; d. placing a first means forviewing incorporating a first Si3N4 thin-film membrane in saidimpression of said second component, said impression approximatelycentered in said second component; e. placing said sample on the centerof said first means for viewing; f. insuring spacing between said firstmeans for viewing an said second means for viewing to accommodate saidsample; g. placing said second means for viewing incorporating a secondSi3N4 thin-film membrane, the second Si3N4 thin-film membrane abuttingsaid sample in said impression, said second means for viewingself-aligning in said impression with said first means for viewing; h.placing a second inner means for sealing in a second slot encirclingsaid Si3N4 thin-film membrane in said first component; i. placing anouter means for sealing in a third slot in said second component, saidouter means for sealing encircling said impression of said secondcomponent; j. placing said first component over said second componentwith captured said second inner and said outer inner means for sealing,aligning said means for fastening in said second component with fourslots in said first component; k. rotating said first component to alignit under said means for fastening; l. establishing a non-fluid flowingchamber by tightening said means for fastening, m. placing said sampleholding device in proximity to a viewing device and indexing saidholding device with said viewing device, and n. repeating steps athrough m for each additional said sample.
 15. The method of claim 14providing said sample as a wet sample.
 16. The method of claim 15providing said wet sample at a thickness of about 50 nm or less.
 17. Themethod of claim 14 providing said viewing means as silicon wafers. 18.The method of claim 17 providing each said silicon wafer at a thicknessof about 200 μm, wherein said transparent membranes are fabricated insaid silicon wafer by at least etching said silicon wafer.
 19. Themethod of claim 14 providing said transparent membranes as thin-filmSi₃N₄-coated membranes at less than about 100 nm thick.
 20. The methodof claim 14 has been changed to—providing said fastening means asmachine screws.